Global Phylogeography and Genomic Epidemiology of Carbapenem-Resistant blaOXA-232–Carrying Klebsiella pneumoniae Sequence Type 15 Lineage

Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has compromised antimicrobial efficacy against severe infections worldwide. To monitor global spread, we conducted a comprehensive genomic epidemiologic study comparing sequences from 21 blaOXA-232–carrying CRKP isolates from China with K. pneumoniae sequence type (ST) 15 strains from 68 countries available in GenBank. Phylogenetic and phylogeographic analyses revealed all blaOXA-232–carrying CRKP isolates belonged to ST15 lineage and exhibited multidrug resistance. Analysis grouped 330 global blaOXA-232–carrying ST15 CRKP strains into 5 clades, indicating clonal transmission with small genetic distances among multiple strains. The lineage originated in the United States, then spread to Europe, Asia, Oceania, and Africa. Most recent common ancestor was traced back to 2000; mutations averaged ≈1.7 per year per genome. Our research helps identify key forces driving global spread of blaOXA-232–carrying CRKP ST15 lineage and emphasizes the importance of ongoing surveillance of epidemic CRKP.

Prevalence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has compromised antimicrobial efficacy against severe infections worldwide.To monitor global spread, we conducted a comprehensive genomic epidemiologic study comparing sequences from 21 bla OXA-232carrying CRKP isolates from China with K. pneumoniae sequence type (ST) 15 strains from 68 countries available in GenBank.Phylogenetic and phylogeographic analyses revealed all bla OXA-232 -carrying CRKP isolates belonged to ST15 lineage and exhibited multidrug resis-tance.Analysis grouped 330 global bla OXA-232 -carrying ST15 CRKP strains into 5 clades, indicating clonal transmission with small genetic distances among multiple strains.The lineage originated in the United States, then spread to Europe, Asia, Oceania, and Africa.Most recent common ancestor was traced back to 2000; mutations averaged ≈1.7 per year per genome.Our research helps identify key forces driving global spread of bla OXA-232 -carrying CRKP ST15 lineage and emphasizes the importance of ongoing surveillance of epidemic CRKP.
carbapenem resistance and many coexist with virulence plasmids (6,9,10).Applying a previously proposed reverse genomic epidemiology strategy enabled us to make genome comparisons of isolates to identify shared sources of infection on the basis of genomic similarity (11).This approach, developed in response to increasing rates of global spread of antimicrobial-resistant bacteria, emphasizes the need to explore transmission of infection from a global rather than a country-or case-specific perspective.Limited data from previous studies on the number and geographic diversity of bla OXA-232 -carrying K. pneumoniae isolates has impeded full understanding of the genomic evolution and transmission dynamics of CRKP (5,12).
We aimed to perform a multicenter molecular epidemiologic study of carbapenem-resistant bla-OXA-232 -carrying K. pneumoniae ST15 isolates in China, focusing on the genomic characterization of the lineage and the genetic context of the bla OXA-232 -carrying plasmids.We performed large-scale comprehensive genomic epidemiologic analysis to investigate the transmission histories, common ancestors, evolution rates, and population structures of all publicly available bla OXA-232 -carrying K. pneumoniae of ST15 lineage.Our study provides data that will help monitor global spread and the epidemic expansion of K. pneumoniae ST15 lineage and provide insights for developing new infection control strategies.The ethics committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, approved this study (2022-0227).

Plasmid Conjugation
We used rifampin-resistant E. coli EC600 as the recipient.We selected transconjugants on Mueller-Hinton agar supplemented with rifampin (300 mg/L) and imipenem (4 mg/L).We counted numbers of transconjugant colonies after overnight incubation at 37°C and confirmed transconjugants using PCR and Sanger sequencing.
After conducting in silico MLST analysis and detecting the presence of bla OXA-232 gene in K. pneumoniae, we selected 330 bla OXA-232 -carrying K. pneumoniae ST15 strains for further investigation.On the basis of geographic information for each isolate, we created a minimum-spanning tree based on core genome MLST allelic profiles of K. pneumoniae ST15 isolates using chewieSnake (23) and visualized the phylogenetic tree using GrapeTree (24).We considered isolates to be closely related genotypically when separated by a distance of ≤20 alleles.We used Snippy version 4.6.0(https://github.com/tseemann/snippy) to align sequences and identify single-nucleotide polymorphisms (SNPs).We parsed genome alignment through Gubbins (https://github.com/nickjcroucher/gubbins), which identified and removed recombination regions, and inferred and constructed a maximum-likelihood phylogeny from those SNPs.We calculated pairwise SNP distances between the genomes of each isolate to define clades.

Phylodynamic Analysis
We performed root-to-tip regression analysis using TempEst version 1.5.3 (https://github.com/beastdev/Tempest) to confirm that the maximum-likelihood tree had sufficient temporal signal.We used a Bayesian Markov Chain Monte Carlo approach implemented in BEAST2 (25) to analyze alignment of putative substitution mutations identified by Gubbins, on which estimates the time-scaled phylogenetic tree was based, as well as time of most recent common ancestor (tMRCA) and mutation rates.We allowed each run 300 million iterations, sampled from every 20,000th iteration, and discarded the first 10% of the samples as burn-in.We estimated the effective sample size using Tracer version 1.7.1 (https://github.com/beast-dev/tracer/releases) to evaluate the operation convergence.We used RhierBAPS version 1.0.1 (26) to analyze the allele frequency parameters of the bacterial population and cluster individual sequences to identify the population structure, then used the resulting clades as input for the SkyGrowth package (https://github.com/mrc-ide/skygrowth) to evaluate effective population sizes.We visualized and annotated phylogenetic trees using the interactive Tree of Life (iTOL) V5 web server (27).We analyzed and visualized transmission links for isolates and nodes with spatial phylogenetic reconstruction of evolutionary dynamics using SpreaD3 (28) under a discrete trait model.We deposited genome sequences of the 21 CRKP isolates in GenBank (BioProject accession nos.PRJNA818898 and PRJNA745926).

Occurrence and Divergence of ST15 Lineage
We investigated global phylogenetic relationships of 2,118 K. pneumoniae ST15 strains using core genome MLST analysis with a cutoff of 20 alleles to define clonality (Appendix 2 We identified all 21 bla OXA-232 CRKP isolates collected in this study as ST15.We performed phylogenetic analysis between the 21 bla OXA-232 -carrying CRKP isolates and 309 bla OXA-232 -carrying K. pneumoniae ST15 isolates obtained from NCBI (Appendix 2 Table 2).The bla OXA-232 -carrying K. pneumoniae ST15 isolates originated from 10 different countries: China, United Kingdom, Nepal, Netherlands, Oman, Thailand, United States, France, India, and Australia.Evaluation of clonal relatedness by core-genome SNPs revealed that the bla OXA-232 -carrying K. pneumoniae ST15 isolates had an average distance of 29 (range 0-208) SNPs.Phylogenetic analysis using hierarchical Bayesian clustering separated ST15 isolates into In-depth phylogenetic analysis revealed details about origins and mutation rates of bla OXA-232 -carrying K. pneumoniae ST15 isolates.The correlation coefficient and R 2 for the root-to-tip genetic divergence (0.773) compared with time in the TempEst analysis (0.598) indicated a strong linear relationship between accumulated mutations and sampling time, suggesting that enough signal was present to cali-brate a strict clock model.The median molecular clock rate was estimated to be 3.40 × 10 −3 (95% highest posterior density interval [HPD] 3.10-3.69× 10 −3 ) substitutions/site/year, which translates to ≈1.7 mutations/genome/year.The tMRCA for bla OXA-232 -carrying K. pneumoniae ST15 lineage was estimated from the temporal height, which dates to 2000 (95% HPD 1996-2003).Strains from China appeared within a recent time window (after 2015) but have greatly increased, apparently the result of a rapidly expanding epidemic of clonal transmission.

Transcontinental Dissemination of Epidemic ST15 Lineage in the 21st Century
The United States is the likely origin of the ST15 lineage (Figure 5), the offspring of which were introduced into other continents, including Europe, Asia, and Oceania.Transmission from the United States to Europe triggered an epidemic in the United Kingdom.The ST15 lineage was concurrently introduced from the United States to China, where it is the most common strain (93.33%, 308/330 isolates).Epidemic clonal transmissions in China were linked to introductions from the United States (2011-2013), Nepal (2015), and the United Kingdom (2020).Additional global transmission events originated in China and reached the United States, Europe, and Australia.Transmission from China to Australia in 2014 triggered subsequent transmission events from Australia to Europe and Asia.Multiple transmission events occurred from Europe and Australia to Asia in 2015-2016, contributing to complex transmission pathways among countries in Asia.In 2015, strains from Australia reached Oman and subsequently spread to Nepal.In the same year, strains from Nepal spread to Thailand and China.In 2016, strains from Europe were introduced into Thailand and further disseminated to Nepal.After a local outbreak, strains from China spread to India in 2019.

Discussion
Worldwide spread of CRKP has been accompanied by considerable incidence and death, posing a severe threat to public health (29,30).Multiple nosocomial outbreaks of bla OXA-232 -carrying K. pneumoniae have occurred in China and may have contributed to the prevalence of those strains (5,6,9,31).We analysed the genomic features of 21 CRKP ST15 isolates carrying bla OXA-232 collected from various regions in China.We integrated the results with global data to investigate the mode of spread and the possible origins of ST15 bla OXA-232 -carrying CRKP.Our findings provide new insights into the genomic characteristics of bla OXA-232carrying K. pneumoniae and transmission dynamics.
Our analysis of bla  worldwide (6,8,13).Our unsuccessful plasmid conjugation experiment demonstrated the presence of bla OXA-232 within a nonconjugative plasmid, confirming findings from several previous studies (7,31).Integration of the bla OXA-232 gene onto IncF-, IncHI1B-, and IncFII-type plasmids through the transposon element ISEcp1 has been documented.IncF-and IncHI1B-type plasmids serve as crucial carriers for multiple antimicrobial resistance genes, such as extended spectrum β-lactamases and bla NDM , that exhibit the ability for interspecies conjugative transfer.Those plasmids are prevalent among members of Enterobacterales and increase risk for wide dissemination (32,33).The integration of ColKP3-type miniplasmid on a larger plasmid suggests that, although not self-transmissible, ColKP3 can be mobilized with the assistance of a self-transmissible plasmid.Of note, plasmids encoding bla OXA-232 are distributed already in South Asia, Europe, and South from China and Nepal based on pairwise distances ≤20 SNPs between these strains.Multiple nosocomial outbreaks of bla OXA-232 -CRKP ST15 across different geographic regions also provided epidemiologic evidence to support our investigation (5,6,9).We also collected 1 K. pneumoniae isolate KP81, carrying both bla OXA-232 and bla KPC-2 , also reported in the United States in 2020 (34).Emergence of CRKP ST15 carrying both bla OXA-232 and bla KPC-2 in China poses a substantial threat to public health because of its potential for spreading carbapenem resistance internationally through the high-risk clone ST15.bla OXA-232 -carrying K. pneumoniae showed low resistance to imipenem and meropenem in our investigation, also described elsewhere (6,9), possibly attributable to disruption of efficient hydrolytic activity because of loss of a salt bridge between residues D159 and R214 (35).We used Bayesian phylogeny to elucidate the evolutionary history of K. pneumoniae carrying bla-OXA-232 within the ST15 lineage.We estimated that this specific lineage appeared in 2000 (95% HPD interval 1989-1998), approximately the same time the ST147 KL10-O3a (2002) and ST258 (1997) lineages were reported (36,37).On the basis of our analysis, we suggest that the ST15 lineage originated in the United States.In addition, we successfully reconstructed early transmission events from the United States to both Europe and Asia.Importated strains from the United States and Nepal to China catalyzed clonal spread of strains already circulating in China.From China, strains of ST15 lineage were disseminated across multiple continents and eventually became globally dispersed.Introduction and inappropriate use of fourth-generation cephalosporin (38) and carbapenem antimicrobial drugs (39,40), approved in clinical settings in the 1990s, may have influenced the global emergence and transmission of ST15 lineage.
Our study was limited by focusing solely on ST15 bla OXA-232 -carrying CRKP strains, which we did because those strains have the highest isolation rate in China and have caused multiple nosocomial outbreaks.Future studies are needed to explore global  distribution of other lineages.Second, we might have underestimated prevalence of the bla OXA-232 gene because of limited carbapenem hydrolysis activity, possibly resulting in false-negative results in antimicrobial susceptibility testing.Finally, although we comprehensively evaluated all publicly available ST15 bla OXA-232 -carrying CRKP strains worldwide, it is possible that some circulating strains went undetected in some regions with dense strain distribution where isolates were derived from a small number of surveillance sites.Therefore, genomic surveillance should be expanded, especially in countries with low isolation rates, to provide a more comprehensive understanding of the emergence, expansion, and spread of CRKP.In conclusion, our research contributes to the comprehension of the global spread of bla OXA-232 -carrying CRKP ST15 lineage and emphasizes the need to develop measures for preventing and controlling progression of the CRKP epidemic.

Figure 1 .
Figure 1.Genetic context of bla OXA-232 identified in patients in China investigated in study of global phylogeography and genomic epidemiology of bla OXA-232 -carrying carbapenem-resistant Klebsiella pneumoniae sequence type 15.Arrows represent coding sequences (red arrows, antimicrobial resistance genes; yellow or blue arrows, mobile elements) and indicate the direction of transcription.Arrow size is proportional to gene length.GenBank accession numbers are provided.

Figure 3 .
Figure 3. Minimum spanning tree based on core genome MLST analysis of global bla OXA-232 -carrying carbapenem-resistant Klebsiella pneumoniae sequence type 15 isolates.Core genome multilocus sequence typing scheme with clade alert distance set as ≤20 alleles.Line lengths connecting each circle depict clonal relationships between isolates.Colors of circles indicate different countries.Numbers in square brackets in the key indicate numbers of isolates recovered from each country.Scale bar represents genetic distance (allelic differences).

Figure 4 .
Figure 4. Phylogenetic analysis of global bla OXA-232 -carrying carbapenem-resistant Klebsiella pneumoniae sequence type (ST) 15 isolates.A) Time-calibrated maximum clade credibility Bayesian phylogeny based on 330 bla OXA-232 -carrying ST15 recombination-filtered core genomes and distribution of antimicrobial resistance genes and virulence genes in the isolates.The cells with colors indicate presence of the gene; blank cells indicate absence.Different colored circles indicate the geographic location and separation time of strains.Blue bars along branches indicate 95% highest posterior probabilities.B) Effective population size of ST15 lineage strains based on the population structure.Shaded areas indicate 95% highest posterior probabilities.Scale bar indicates number of base substitutions per site.

Figure 5 .
Figure 5. Temporal and spatial transmission trajectory of global bla OXA-232 -carrying carbapenem-resistant Klebsiella pneumoniae.Coralcolored countries on map indicate geographic regions where bla OXA-232 has occurred; arrows show dates and direction of transmission.The bla OXA-232 -carrying isolate originated in the United States, initially expanded to the United Kingdom and China, then spread to the rest of the world, with China as its central focus.Red circles represent major outbreak regions in the United Kingdom and China; size of the red circles corresponds to the number of strains analyzed in each country.

Table 1 .
Characteristics of 21 infected patients in China investigated in study of global phylogeography and genomic epidemiology of blaOXA-232-carrying carbapenem-resistant Klebsiella pneumoniae sequence type 15
OXA-232 -carrying CRKP genomes found bla OXA-232 on a 6.1 kb ColKP3 plasmid in isolate KP3295 with high similarity and coverage with previously reported plasmids carrying bla OXA-232 detected 2252Emerging Infectious Diseases • www.cdc.gov/eid• Vol. 29, No. 11, November 2023 This study was supported by the National Natural Science Foundation of China (82102436, 81871696, 82202565), the Zhejiang Provincial Natural Science Foundation of China (LR23H200001, LY21H190001, LQ22H200001), and the Zhejiang Provincial Medical and Health Science and Technology Plan (2020RC066, 2021RC007, 2022RC299, 2023KY227, 2023KY228).Ms. Wu is a graduate student at Zhejiang University School of Medicine.Her research interests include molecular epidemiology and antimicrobial resistance mechanisms of MDR bacterial pathogens.Dr. Ruan is a principal investigator at Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, China.His primary research interests are microbial genomics and application of bioinformatics to study the genomic epidemiology of multidrug-resistant bacterial infections.